Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/02/12/jpet.115.231647.DC1 1521-0103/357/1/157–166$25.00 http://dx.doi.org/10.1124/jpet.115.231647 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 357:157–166, April 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Stronger Uricosuric Effects of the Novel Selective URAT1 Inhibitor UR-1102 Lowered Plasma Urate in Tufted Capuchin Monkeys to a Greater Extent than Benzbromarone s Sung Oh Ahn, Shuichi Ohtomo, Jumpei Kiyokawa, Toshito Nakagawa, Mizuki Yamane, Kyoung June Lee, Ki Hwan Kim, Byung Ho Kim, Jo Tanaka, Yoshiki Kawabe, and Naoshi Horiba Discovery Research Center, C&C Research Laboratories, Suwon, Republic of Korea (S.O.A., B.H.K.); Research Division, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka, Japan (S.O., J.K, T.N., M.Y., Y.K., N.H.); Drug Discovery Center, JW Downloaded from Pharmaceutical Corp. Seoul, Republic of Korea (K.J.L.); JW CreaGene, Seongnam, Republic of Korea (K.H.K.); Drug Safety Research Laboratory, Shin Nihon Biological Laboratories, Miyanoura, Kagoshima, Japan (J.T.) Received December 21, 2015; accepted February 11, 2016 ABSTRACT jpet.aspetjournals.org Urate-lowering therapy is indispensable for the treatment of gout, or benzbromarone at 3–100 mg/kg was administered orally once a but available drugs do not control serum urate levels tightly enough. day for 3 consecutive days to tufted capuchin monkeys, whose low Although the uricosurics benzbromarone and probenecid inhibit a uricase activity causes a high plasma urate level. When compared urate reabsorption transporter known as renal urate transporter with the same dosage of benzbromarone, UR-1102 showed a 1 (URAT1) and thus lower serum urate levels, they also inhibit other better pharmacokinetic profile, increased the fractional excretion of transporters responsible for secretion of urate into urine, which urinary uric acid, and reduced plasma uric acid more effectively. suggests that inhibiting URAT1 selectively would lower serum urate Moreover, the maximum efficacy of UR-1102 was twice that of more effectively. We identified a novel potent and selective URAT1 benzbromarone, suggesting that selective inhibition of URAT1 is at ASPET Journals on September 30, 2021 inhibitor, UR-1102, and compared its efficacy with benzbromarone effective. Additionally UR-1102 showed lower in vitro potential for in vitro and in vivo. In human embryonic kidney (HEK)293 cells mechanisms causing the hepatotoxicity induced by benzbromarone. overexpressing URAT1, organic anion transporter 1 (OAT1), and These results indicate that UR-1102 achieves strong uricosuric OAT3, benzbromarone inhibited all transporters similarly, whereas effects by selectively inhibiting URAT1 over OAT1 and OAT3 in UR-1102 inhibited URAT1 comparably to benzbromarone but monkeys, and could be a novel therapeutic option for patients inhibited OAT1 and OAT3 quite modestly. UR-1102 at 3–30 mg/kg with gout or hyperuricemia. Introduction be lowered sufficiently to improve signs and symptoms of gout, with a target minimum of 6 mg/dl, or in many cases 5 mg/dl Gout has become a global burden in recent years because of (Khanna et al., 2012). Moreover the lower the serum urate level, its increasing prevalence, extensive comorbidity associated the faster and more significant the reduction in the size of tophi, with disability, and increased mortality (Robinson and Dalbeth, which are solid deposits of monosodium urate crystals (Perez- 2015). Because the clinical manifestations of gout are associ- Ruiz et al., 2002, 2007). This is ordinarily achieved with either a ated with the formation of monosodium urate crystals in the urate synthesis inhibitor (USI) or a uricosuric (Shahid and joints, controlling serum urate is important when treating Singh, 2015). The ACR guidelines recommend the use of a USI, gout. Several major guidelines, including the 2012 American College of Rheumatology Guidelines for Management of Gout such as allopurinol or febuxostat, as the first-line approach for (ACR guidelines), recommend that serum urate level should pharmacologic urate-lowering therapy and, because the urate- lowering efficacy of currently available USI drugs is insufficient, that this be combined with a uricosuric when the serum urate Primary laboratory of origin: C&C Research Laboratories. Results of this study were presented in part as: Ahn SO, Horiba N, Ohtomo target is not met by the USI. Typical uricosurics, namely S, Lee KJ, Kim KH, Kim BH, Kiyokawa J, Yamane M, Ikegami H, Nakagawa benzbromarone or probenecid, increase the urate excretion by T, et al. (2013) The therapeutic efficacy of the novel uricosuric agent UR-1102 inhibiting urate transporter 1 (URAT1), the transporter re- for hyperuricemia and gout. Annual European Congress of Rheumatology; June 2013; Madrid, Spain. Abstr. no. SAT0356, The European League Against sponsible for urate reabsorption in the renal proximal tubules Rheumatism (EULAR), Kilchberg, Switzerland; published as Ann Rheum Dis (Enomoto et al., 2002; Shin et al., 2011). 72 (suppl. 3):704. — dx.doi.org/10.1124/jpet.115.231647. URAT1 is a member of the SLC22A or organic anion s This article has supplemental material available at jpet.aspetjournals.org. transporter (OAT)—family, and its mutations can cause ABBREVIATIONS: FEUA, fractional excretion of uric acid; HEK, human embryonic kidney; LDH, lactate dehydrogenase; MEM, minimum essential medium; MMP, mitochondrial membrane potential; OAT, organic anion transporter; PAH, p-aminohippuric acid; PK, pharmacokinetic; URAT1, urate transporter 1; USI, urate synthesis inhibitor. 157 158 Ahn et al. hypouricemia (Ichida et al., 2004; Dinour et al., 2011). pyrido-oxazine. 4H-Pyrido[4,3-b][1,4]oxazin-3-one was converted Transporters in the SLC22A family play important roles in to the corresponding pyrido-oxazine by reaction with 1.0 mol/l the reabsorption and excretion of anionic substances, includ- lithium aluminum hydride. The resulting pyrido-oxazine was then ing urate and other endogenous substances, xenobiotics, and reacted with 3,5-dibromo-4-methoxybenzoyl chloride, followed by drugs in the kidney (Burckhardt, 2012). In addition to URAT1, demethylation under acidic conditions. Details of synthesis are included in Example 4 in Ahn et al. (2013). [14C]UR-1102 and [14C] OAT1, and OAT3 are also SLC22A family members and trans- benzbromarone were synthesized at Korea RadioChemicals Center port urate in the kidney; however, these are thought to be (Suwon, Republic of Korea). Benzbromarone was purchased from responsible for the secretion of urate into the urine, whereas Sigma-Aldrich/MerckMillipore (St. Louis, MO). [8-14C]Uric acid ([14C] URAT1 is responsible for reabsorption (Bakhiya et al., 2003; uric acid) and [ring-14C]acetamidophenol ([14C]acetaminophen) were Ichida et al., 2003; Choi et al., 2005). In fact, inhibition of OAT3 purchased from American Radiolabeled Chemicals (St. Louis, MO). is thought to be one reason why some angiotensin II receptor p-[Glycyl-2-3H]-aminohippuric acid ([3H]PAH) was obtained from blockers, such as candesartan or valsartan, can cause hyper- PerkinElmer Inc. (Waltham, MA). p-Aminohippuric acid (PAH) and uricemia (Sato et al., 2008). Probenecid, which is considered acetaminophen were purchased from Wako Pure Chemical Industries, to have insufficient clinical efficacy in lowering urate is Ltd. (Tokyo, Japan). All other chemicals were of the highest purity known to inhibit not only URAT1 but also OAT1 and OAT3 available. (Takeda et al., 2001; Shitara et al., 2005; Kusuhara et al., 2013). Although the precise contribution of the two secretory In Vitro Transporter Inhibition Assays transporters to urate excretion remains unclear, selective Downloaded from Cell Culture and Transfection. An expression plasmid contain- inhibition of URAT1 would potentially achieve stronger ing human SLC22A12 (URAT1) (GenBank Accession No. NM_144585) urate excretion. Benzbromarone lowers serum urate more was prepared by digesting the fragments from URAT1-pME18SFL3 effectively than probenecid; however, it is only available in a vector (Toyobo Co., Tokyo, Japan) and ligating them into the mul- few countries outside the United States, owing to safety ticloning site of pcDNA3.1(1) (Life Technologies/Thermo Fisher concerns, which include the risk of hepatitis (Lee et al., Scientific, Waltham, MA). Expression plasmids containing human 2008). In addition, benzbromarone also reportedly inhibits SLC22A6 (OAT1) and SLC22A8 (OAT3) (GenBank Accession Nos. jpet.aspetjournals.org OAT3 (Kimura et al., 2000), which suggests that its urico- NM_153276, NM_004254) were purchased from Origene Technologies suric efficacy could be surpassed by an inhibitor that is (Rockville, MD). selective only for URAT1. In summary, a safe and potent Human embryonic kidney (HEK)293 cells (American Type Culture Collection, Manassas, VA), a transformed cell line de- uricosuric drug that can achieve target serum urate levels of rived from human embryonic kidney, were cultured in complete less than 5 mg/dl is required to treat patients with gout, medium consisting of Dulbecco’s modified Eagle’smedium especially those with tophi, and a selective URAT1 inhibitor (Sigma-Aldrich/MerckMillipore) with 10% fetal calf serum in an at ASPET Journals on September 30, 2021 could be one approach for realizing this concept. atmosphere of 5% CO2, and 95% air at 37°C. Cells were seeded in It is well known that there are